Two isoforms of ferredoxin:NADP+ oxidoreductase from wheat leaves: purification and initial biochemical characterization

Abstract

Ferredoxin:NADP(+) oxidoreductase is an enzyme associated with the stromal side of the thylakoid membrane in the chloroplast. It is involved in photosynthetic linear electron transport to produce NADPH and is supposed to play a role in cyclic electron transfer, generating a transmembrane pH gradient allowing ATP production, if photosystem II is non-functional or no NADP(+) is available for reduction. Different FNR isoforms have been described in non-photosynthetic tissues, where the enzyme catalyses the NADPH-dependent reduction of ferredoxin (Fd), necessary for some biosynthetic pathways. Here, we report the isolation and purification of two FNR isoproteins from wheat leaves, called FNR-A and FNR-B. These forms of the enzyme were identified as products of two different genes, as confirmed by mass spectrometry. The molecular masses of FNR-A and FNR-B were 34.3 kDa and 35.5 kDa, respectively. The isoelectric point of both FNR-A and FNR-B was about 5, but FNR-B appeared more acidic (of about 0.2 pH unit) than FNR-A. Both isoenzymes were able to catalyse a NADPH-dependent reduction of dibromothymoquinone and the mixture of isoforms catalysed reduction of cytochrome c in the presence of Fd. For the first time, the pH- and ionic strength dependent oligomerization of FNRs is observed. No other protein was necessary for complex formation. The putative role of the two FNR isoforms in photosynthesis is discussed based on current knowledge of electron transport in chloroplasts.

Identification of osmotic stress-responsive genes from <i>Leymus mollis</i>, a wild relative of wheat (<i>Triticum aestivum</i> L.)

“…ABC transporters participate in chlorophyll biosynthesis, stomatal movement, ion influx and detoxification processes (Martinoia et al 2002). We also identified an EST for ferredoxin-NADP(H) oxidoreductase (FNR; JK317158), which catalyzes the final step in the photosynthetic linear electron transfer chain in chloroplasts (Grzyb et al 2008). FNR transports electrons from reduced ferredoxin (Fd) to NADP + to form NADPH.…”

Ferredoxin:NADP+ oxidoreductase bound to cytochrome b6f complex is active in plastoquinone reduction: Implications for cyclic electron transport

“…All the investigated preparations of the complex showed the presence of FNR polypeptide – with one or two isoforms of the enzyme as revealed by immunoblot analysis. In the literature, there are several reports on the identification of leaf isoenzymes of FNR in Arabidopsis (Lintala et al 2007), wheat (Grzyb et al 2008), rice and maize (Okutani et al 2005). In the latter species, three leaf isoenzymes have been identified (Okutani et al 2005), of which two were co‐purified with the cytochrome b 6 f complex.…”

Energy Conductance from Thylakoid Complexes to Stromal Reducing Equivalents

“…The chloroplast FNR proteins seem to be at least partly redundant, but they also possess unique properties, which are probably required for adjustment of chloroplast metabolism according to changes in the ambient environment (Mulo, 2011). FNR gene families have been well studied in the dicot C 3 plant Arabidopsis thaliana (thale cress, Hanke et al, 2005), monocot C 3 plant Triticum aestivum (wheat, Gummadova et al, 2007;Grzyb et al, 2008), and monocot C 4 plant Zea mays (maize, Okutani et al, 2005). In Arabidopsis two genes, At5g66190 and At1g20020, encode the two distinct ~ 32 kDa leaf isoforms FNR1 and FNR2 Lintala et al, 2007).…”

“…It is very likely that FNR in vivo exists as a (hetero)dimer . Recently, also formation of large (~ 330 kDa) FNR oligomers, devoid of other proteins, has been documented (Grzyb et al, 2008). Inactivation of one chloroplast FNR isoform did not result in upregulation of the expression of the other, neither at the level of transcription nor translation (Lintala et al, 2007;Lintala et al, 2009).…”

“…Based on isoelectric focusing and SDS-PAGE the proteome of wheat has revealed four distinct leaf FNR isoforms that can be divided into two groups, FNRI and FNRII. Both groups contain two proteins, which differ from each other by truncation of the N-terminus (Gummadova et al, 2007;Grzyb et al, 2008). It has been demonstrated that the presence of mature wheat FNR proteins with alternative N-terminal start points, differing by a three amino acid truncation in pFNRI and a two amino acid truncation in pFNRII, have statistically significant differences in response to the physiological parameters of chloroplast maturity, nitrogen regime, and oxidative stress (Moolna et al, 2009).…”